OVERPRODUCTION AND RAPID PURIFICATION OF HUMAN FAST SKELETAL BETA-TROPONIN-T USING ESCHERICHIA-COLI EXPRESSION VECTORS - FUNCTIONAL DIFFERENCES BETWEEN THE ALPHA-ISOFORM AND BETA-ISOFORM

Troponin T (TpnT), an essential component of the Ca2+-regulatory troponin complex, is involved in protein-protein interactions with other thin-filament proteins during muscle contraction in vertebrate striated muscle (VSM). The isoforms of TpnT are encoded by members of a multigene family which, by alternate splicing, produces a complex pattern of isoproteins in VSM, The functional domains of TpnT are only tentatively identified and structure-function analysis on this protein is limited due to the heterogeneity of the multiple isoforms. We reasoned that the overproduction and purification of a single TpnT species in Escherichia coli would provide an insight into these studies, besides being useful in crystallizing the protein. We cloned the human fast skeletal beta TpnT-encoding cDNA (beta TpnT(f)) in three expression vectors. Overexpression was achieved in an E. coli BL21(DE3) lysogen using a T7 RNA polymerase promoter-based vector, pET17b. The unfused recombinant protein was purified by a simple and rapid procedure in a biologically active and immunoreactive form. This is the first successful synthesis of a complete beta TpnT(f) polypeptide from any species using an in vitro expression system. Purified human beta TpnT(f), a predominant fetal form, was less Ca2+-sensitive and exhibited considerably reduced affinity for troponin C and tropomyosin, as compared to the rabbit fast skeletal alpha TpnT, a predominant adult isoform, These results provide a biochemical correlate to the age-related differences in Ca2+ sensitivity of tension development in vertebrate fast skeletal muscles.